Unexpected Gelators with Unforeseen Properties: A Spectroscopic Investigation of Alanine-Based Self-Assembling Hydrogels

Abstract

Self-assembly of biomolecules is a prominent issue explored in biomedical, biophysical, and biomaterial research. Understanding how and why certain peptides/proteins prefer to self-assemble into larger networks can reveal the mechanism of amyloid formation and assist in bottom-up designs of supramolecular structures like gels and nanotubes. Hydrogels formed by polypeptides could be much-favored tools for drug delivery and other biotechnical applications due to their main ingredients being biodegradable. However, the gelation of peptides in aqueous solution is generally thought to require a minimal length of the peptide chain, as well as distinct sequences of hydrophilic and hydrophobic residues. This biodegradability and cheap manufacturing costs are increased for shorter peptides, but a high degree of hydrophobicity or aromaticity was deemed necessary. Contrary to expectation, it was discovered that cationic glycyl-alanyl-glycine (GAG) in ethanol/water mixtures forms a gel comprised of network spanning fibrils several 100 μm long. To explore how ethanol solvation and solvent-peptide interactions prepare the system for aggregation and fibrillization, we utilized amide I band profiles along with J-coupling constants and chemical shift from NMR measurements to observe conformational changes of the alanine residue caused by the addition of ethanol. We propose that GAG accumulates on the ethanol/water interface, which would increase the effective chemical potential of the peptide and thus would facilitate its self-assembly. We first focused our investigation of the gelation phase on 220 mM GAG in 55 mol% ethanol/45 mol% water. Rheology shows that under optimal conditions, the storage modulus is 106 Pa. UVCD, VCD and IR measurements, supported by DFT calculations, suggested that the underlying structure of the fibrils is dependent on the formation temperature of the fibrils and that it is not solely comprised of β-sheets. It was determined through annealing cycles that the fibrillar aggregates are not the thermodynamically preferred structure. Instead, amorphous aggregates were formed and stable. At short incubation times, the reformed gels are weakened suggesting the strength of the gel and melting temperature can be easily tuned. A phase diagram was constructed by systematically adjusted the amount of GAG dissolved into different ethanol/water mixtures. We observed that the gel phase is stabilized by increasing the ethanol fraction more so than by an increase of the peptide concentration. UVCD was utilized to determine the melting temperature of all formed gels to add a third dimension to the phase diagram.Ph.D., Chemistry -- Drexel University, 201